1. Field of the Invention
This invention pertains to the field of treating inflammation. Compounds and methods for modulating an inflammatory response without significantly affecting lymphocyte trafficking are provided.
2. Background
A mammal often responds to cell injury, infection, or an abrupt change in a tissue by inducing an inflammatory response. Typically, an inflammatory response is initiated by endothelial cells producing molecules that attract and detain inflammatory cells (e.g., myeloid cells such as neutrophils, eosinophils, and basophils) at the site of injury. The inflammatory cells then are transported through the endothelial barrier into the surrounding tissue. The resulting accumulation of inflammatory cells, in particular neutrophils, is followed by generation of toxic oxygen particles and, release of neutrophil granules which contain acid hydrolases and degradative enzymes such as proteases, elastase, and collagenase, which contribute to local tissue breakdown and inflammation. Neutrophils can also release chemoattractants and complement activators that amplify the inflammation.
Although the inflammatory response can play a role in the healing process by destroying, diluting, and isolating injurious agents and stimulating repair of the affected tissue, inflammatory responses can also be harmful, and indeed life-threatening. Five symptoms often characterize the inflammatory response: pain, redness, heat, swelling, and loss of function. For example, inflammation results in leakage of plasma from the blood vessels. Although this leakage can have beneficial effects, it causes pain and when uncontrolled can lead to loss of function and death (such as adult respiratory distress syndrome). Anaphylactic shock, arthritis, and gout are among the conditions that are characterized by uncontrolled or inappropriate inflammation.
Inflammatory responses differ from immune responses mediated by T- and B-lymphocytes in that an inflammatory response is non-specific. While antibodies and MHC-mediated immune responses are specific to a particular pathogen or other agent, the inflammatory response does not involve identification of a specific agent. Both inflammatory responses and specific immune responses, however, involve extravasation of the respective cell types from the blood vessels to the site of tissue injury or infection. Moreover, several of the receptors that mediate extravasation of lymphocytes are also involved in extravasation of inflammatory cells. In particular, lymphocyte trafficking to lymph nodes under normal circumstances is mediated by selectins that are expressed by cells of the vascular endothelium in response to cytokine induction. Selectins are also involved in the recruitment of neutrophils to the vascular endothelium during inflammation (reviewed in Kansas (1996) Blood 88: 3259-87; McEver and Cummings (1997) J. Clin. Invest. 100: 485-91). Three types of selectins are involved in the interaction between leukocytes and the vascular endothelium. E-selectin (also called endothelial-leukocyte adhesion molecule-1, ELAM-1) and P-selectin are expressed on activated endothelium. P-selectin is also present on activated platelets, while L-selectin is found on lymphocytes. Selectin deficiencies result in varying degrees of impaired lymphocyte trafficking, reduced neutrophil recruitment to sites of inflammation and decreased leukocyte turnover (Arbones et al. (1994) Immunity 1: 247-260; Johnson et al. (1995) Blood 86: 1106-14; Labow et al. (1995) Immunity 1: 709-720; Mayadas et al. (1993) Cell 74: 541-554).
Binding of leukocytes to selectins is at least partially mediated by oligosaccharide ligands that are displayed the surface of the cells. The oligosaccharide ligands are generally attached to glycoproteins and glycolipids. The types of oligosaccharides that carry the physiologically relevant selectin ligands are largely undefined at present, with a variety of possibilities existing among N-glycans, O-glycans, glycolipids, as well as proteoglycans (reviewed in Varki (1997) J. Clin. Invest. 99: 158-162).
A major ligand for P-selectin is reported to be a protein known as “P-selectin glycoprotein ligand-1” (PSGL-1; Li et al. (1996) J. Biol. Chem. 271: 3255-3264; Norgard et al. (1993) J. Biol. Chem. 268: 12764-12774). PSGL-1 is a membrane mucin that is disulfide-linked dimer of two 120 kDa subunits. Each subunit of PSGL-1 contains about 70 extracellular serine and threonine residues that are potential sites for O-glycosylation and three potential sites for N-glycosylation (Norgard et al., supra.). Site-directed mutagenesis of PSGL-1 supports a role for tyrosine and threonine residues in the adhesion of PSGL-1 to selecting, thus suggesting that an O-linked glycan is involved (Li et al. (1996) J. Biol. Chem. 271: 3255-3264).
Serine/threonine (O)-linked oligosaccharides are diverse structures that are prevalent on cell surfaces and secreted proteins. For example, sulfated, sialylated O-linked oligosaccharides on the high endothelial venules (HEVs) of secondary lymphoid organs are ligands for L-selectin. These ligands are also present on various selectin counter-receptors including CD34 (Baumhueter et al. (1993) Science 262: 436-438, GlyCAM-1 (Lasky et al. (1992) Science 262: 436-438), MAdCAM-1 (Berg et al. (1993) Nature 366: 695-698), Sgp200 (Rosen and Bertozzi (1994) Curr. Opin. Cell Biol. 6: 663-673) and the podocalyxin-like protein (Sassetti et al. (1998) J. Exp. Med. 187: 1965-1975). Other reported selectin counter-receptors in which O-glycosylation does not appear to be essential include E-selectin ligand-1 (ESL-1)(Steegmaier et al. (1995) Nature 373: 615-620), CD24 (Aigner et al. (1995) Int. Immunol. 7: 1557-1565), as well as heparin proteoglycans (Norgard-Sumnicht et al. (1993) Science 261: 480-483). Moreover, glycolipids contain selectin ligands that function in vitro (Alon et al. (1995) J. Immunol. 154: 5356-5366); Larkin et al. (1992) J. Biol. Chem. 267: 13661-8); Stroud et al. (1996) Biochemistry 35: 758-69).
Widespread expression of C2 GlcNAcT activity among most tissues may explain why the majority of mammalian O-glycans are of the core 2 subtype (FIG. 1)(Brockhausen (1995) “Biosynthesis of O-glycans of the N-acetylgalactosamine-α-Ser/Thr linkage type.” In Glycoproteins, Montreull et al., eds. (Elsevier Science) pp. 210-259. Schachter and Brockhausen (1989) Symp. Soc. Exp. Biol. 43: 1-26). Core 2 oligosaccharides are common components of mucins, which are glycoproteins for which the majority of their mass is attributable to O-linked oligosaccharides. Mucins are considered to be essential for respiratory epithelium, the gastrointestinal tract and the immune system, by providing a protective function to cell surfaces and regulating cell-cell interactions (reviewed in Hounsell et al. (1996) Glycoconjugate J. 13: 19-26; Strous and Dekker (1992) Crit. Rev. Biochem. Mol. Biol. 27: 57-92).
Core 2 O-glycans are biantennary and may be diversified by glycosyltransferases that add N-acetylglucosamine (GlcNAc) and galactose (Gal) monosaccharides in β1-3 and β1-4 linkages, thereby generating lactosamine disaccharide repeats termed polylactosamines. These can be further modified with sialic acid (Sia) and L-fucose (Fuc) linked at terminal positions. Such modifications to core 2 O-glycan biosynthesis can provide the oligosaccharide ligands for the selectin family of leukocyte adhesion molecules (FIG. 1)(reviewed in Lasky (1995) Annu. Rev. Biochem. 64: 113-139; Lowe (1997) Kidney Int. 51: 1418-1426; Springer (1995) Annu. Rev. Physiol. 57: 827-72). Both lymphocyte homing and neutrophil recruitment in inflammation require the α1-3 fucosyltransferase VII (FucT-VII) enzyme (Maly et al. (1996) Cell 86: 643-653), thus suggesting that a fucosylated oligosaccharide is involved in selectin adhesion. The FucT-VII enzyme can act on N- and O-linked glycans, as well as on glycolipids.
A key branching enzyme that controls O-glycan structural diversity in the synthesis of mammalian O-linked oligosaccharides (O-glycans) is the core 2 β1-6 N-acetylglucosaminyltransferase (C2 GlcNAcT)(Bierhuizen and Fukuda (1992) Proc. Nat'l. Acad. Sci. USA 89: 9326-9330); Williams and Schachter (1980) J. Biol. Chem. 255: 11247-11252). Kumar et al. reported that transfecting a gene that encodes the core 2 β1-6 N-acetylglucosaminyl-transferase into Chinese hamster ovary (CHO) cells that also express PSGL-1 and the fucosyltransferase resulted in high affinity binding to P selectin (Blood (1996) 88: 3872-3879).
Core 2 O-glycan synthesis is also reportedly involved in regulation of lymphoid cell physiology and immune responses (Tsuboi and Fukuda (1997) EMBO J. 16: 6364-6373). Antigen-mediated activation of peripheral T and B cells is characterized by upregulation of core 2 GlcNAc-T activity and branched O-glycans on CD43 (Baum et al. (1995) J. Exp. Med. 181: 877-887); Pilleretal. (1988) J. Biol. Chem. 263: 15146-15150). Core 2 GlcNAc-T has also been implicated in Wiskott-Aldrich Syndrome (WAS)(Higgins et al. (1991) J. Biol. Chem. 266: 6280-6290); Piller et al. (1991) J. Exp. Med. 173: 1501-1510), AIDS (Fox et al. (1983) J. Immunol. 131: 762-7), and leukemia (Brockhausen et al. (1991) Cancer Res. 51: 1257-1263; Saitoh et al. (1991) Blood 77: 1491-9).
Leukocyte extravasation, lymphocyte trafficking, and other processes thus apparently involve oligosaccharide structures that require core 2 GlcNAc transferase. This has hampered the ability to develop treatments that are effective against chronic and otherwise undesirable inflammation but do not compromise the body's ability to mount an effective defense against pathogens and other agents. Ideally, such treatments would interfere with the inflammatory response at an early stage, while not having an adverse effect on the lymphocyte-mediated immune responses. The present invention fulfills this and other needs.